Transmissible gastroenteritis vaccines and methods of producing the same

ABSTRACT

TRANSMISSILBE GASTROENTERITIS VIRUS IS GROWN AND PROPAGATED IN TISSUE CULTURES BY INOCULATING VIRULENT VIRUS PARTICLES INTO A FIRST TISSUE CULTURE, ALLOWING THE VIRUS TO GROW, INTRODUCING THE VIRAL PARTICLES INTO OTHER TISSURE CULTURES UNTIL A CONTINUATED VIRUS IS OBTAINED. A FINAL VIRUS CULTURE CAN BE HARVESTED AND COMBINED WITH A STABLILIZER AND FURTHER INCUBATED TO INACTIVATE THE VIRUS. SOWS AND THEIR NURSING PIGS CAN BE IMMUNIZED BY INJECTING INTO THE PREGNANT SOW BEFORE FARROWING THE VACCINE THUS PRODUCED.

United States Patent Oifice US. Cl. 195-13 2 Claims ABSTRACT OF THEDISCLOSURE Transmissible gastroenteritis virus is grown and propagatedin tissue cultures by inoculating virulent virus particles into a firsttissue culture, allowing the virus to grow, introducing the viralparticles into other tissue cultures until a continuated virus isobtained. A final virus culture can be harvested and combined with astabilizer and further incubated to inactivate the virus. Sows and theirnursing pigs can be immunized by injecting into the pregnant sow beforefarrowing the vaccine thus produced.

This application is a division of application Ser. No. 671,526, filedAug. 24, 1967, now US. Pat. No. 3,479,- 430, which in turn is a divisionof application Ser. No. 398,398, filed Sept. 22, 1964, and nowabandoned.

This invention relates to the art of immunizing animals againsttransmissible gastroenteritis, herein referred to as T.G.E. Moreparticularly the invention relates to the isolation and propagation ofT.G.E. virus, and to processes of preparing killed and attenuatedvaccines thus prepared and their use in protecting sows and theirnursings pigs and immunologically mature swine.

Transmissible gastroenteritis is a highly infectious and widespreadswine disease which causes serious economic loses. T.G.E. may affectswine of all breeds and all ages, but causes extensive mortality lossesonly in very young pigs. T.G.E. was first described by Doyle andHutchings in 1946 (J.A.V.M.A., vol 108: 257-259), and since that time ithas been reported in many states in this country and in other countriesIt has been the practice in the field to feed infective intestinaltracts of infected pigs to sows three or more weeks before farrowing. Asa result passive immunity is transferred to the baby pigs. Thispractice, however, is unsound in that it serves to further disseminatethe disease. Heretofore, it has not been possible to consistently passthe viral agent in tissue culture and control T.G.E. by immunizing theanimals with tissue culture vaccines. Numerous attempts to isolate andserially propagate T.G.E. virus have failed. The present discovery thatthe T.G.E. virus can be propagated in tissue cultures and a vaccineprepared therefrom is the first of its kind, and, consequently, ofconsiderable commercial importance.

An object of the invention is provision of a novel method of initiatinggrowth of T.G.E. virus, and propagating it in tissue cultures.

Another object is the provision of fluids With a high content of T.G.E.viral particles in highly purified form free from excessive cellulardebris which can produce untoward reactions in vaccinated animals. 7

An additional object is the provision of novel inactivated or attenuatedvaccines containing virus produced in tissue cultures which willimmunize sows, thereby protecting them and their offspring while theyare nursing and all other swine, which are immunologically mature,against T.G.E.

A further object is the provision of a novel method of attenuatingT.G.E.

3,585,108 Patented June 15, 1971 Still further objects and the entirescope of applicability of the invention will become apparent from thedetailed description given hereinafter. It should be understood,however, that the detailed description and specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

It has nOW been found that these objects can be attained by the use ofthe following methods. The growth of the T.G.E. virus is initiated andthe virus is propagated in tissue cultures of bovine, porcine, canine(e.g. dog or fox), feline (eg. cat), ferret, ovine and other animaltissues. The propagation can be in the same or different tissues thanthose employed in the first passage.

Attenuation of T.G.E. virus is accomplished by propagation of the virusin tissue cultures at intervals of 24 hours or less until the virus nolonger produces symptoms of T.G.E. in baby pigs. The virus is seriallypassed at such intervals of 24 hours or less until the virus isnon-pathogenic and a vaccine prepared therefrom will stimulateimmunogenesis in swine without producing symptoms of T.G.E. and withoutspreading foreign viruses.

Preferably kidney tissues are employed in making the tissue culturesalthough it should be understood that other tissues can also be used.

After initiation of the growth of the T.G.E. virus and propagationthereof a vaccine is prepared by harvesting the virus containingcultures, preferably adding a stabilizer and then incubating the vaccineto inactivate the T.G.E. virus. The vaccine can be stored as a liquid orit can be frozen, e.g. it can be freeze dried.

The inactivated T.G.E. vaccine is used to immunize sows and theirnursing pigs against T.G.E. by injecting the sow before farrowing withthe vaccine to stimulate the production of antibodies which protects thesow, and at the same time are passed in the milk, thereby protecting thenursing pigs. The sows can be injected intramuscularly orsubcutaneously.

Unless otherwise indicated all parts and percentages are by volume.

EXAMPLE 1 The original starting virus was obtained from baby pigsinfected with T.G.E. The intestinal tracts of infected pigs werestripped of their mucosa. The mucosa was then diluted with a bufferedsalt solution, supplemented with horse serum and homogenized. Thebuffered salt solution was composed of 16 grams KH PO 34 grams of NaHPO, and 8.5 parts of NaCl in 1000 ml. of aqueous solution. One part ofhorse serum by volume was added to nine parts of the salt solution. Onepart of mucosa was added to four parts by volume of the complete saltsolution. This buffered salt solution is entirely different than HanksBalanced Salt Solution and other salt solutions conventionally used fortissue culture nutrient fluids.

The homogenate was centrifuged and passed through a Seitz filter torender it bacteriologically sterile. All of the above steps were carriedout at 0 to 4 C.

The filtrate prepared in Example 1 was then added to various tissueculture systems, monolayer or suspension which was prepared according tocommonly used procedures. The medium generally employed was 9 to 9.5parts by volume of Hanks Balanced Salt Solution (Hanks, J. Cell Comp.Physical, vol. 31, pages 235-260, 1948), and 0.5 to 1 part by volume ofinactivated horse serum. Other media which are satisfactory forinitiating and maintaining infected cultures include (a) Earles BSS plus510% horse serum and (b) Medium 199 of Morgan et al. (Proc. Soc. Exp.Biol. and Med., vol. 73', pages 18,

1959) supplemented with -10% horse serum. Optionally lactalbuminhydrolysate in a final concentration of 0.5% can be added to the abovementioned media. In propagating and attenuating the virus in accordancewith this invention, any nontoxic nutrient fluid tissue culture mediumcan be utilized. In the following examples the medium employed was 9parts of Hanks Balanced Salt Solution and 1 part inactivated horse serum(by volume).

The pH of the medium was adjusted to 7.2 to 7.6 with incubation of thecultures at 35 to 38 C. Serial passage of T.G.E. virus in tissuecultures was carried out at intervals of 24 hours or less, mostfrequently at 6 to 14 hours, by inoculating fresh tissue cultures withpooled diluted fluids from the previous passage. Harvested and pooledfluids were also orally inoculated into baby pigs at every fifth passagelevel to check for presence and/or survival of the virus.

T.G.E. virus virulent for baby pigs was present in titers of at leastafter 5 to passages. Isolation and propagation of T.G.E. virus in tissueculture has been accomplished from different pigs infected with T.G.E.In each case the presence of virus was demonstrated by oral inoculationof baby pigs which developed clinical symptoms of T.G.E. and died. Inaddition, the presence of virus was demonstrated by electron microscopicexamination of tissue culture fluids. Virus particles observed in tissueculture fluids were identical to virus particles isolated fromintestinal mucosal extracts of infected baby pigs.

Electron microscopic identification of this virus as T.G.E. wasaccomplished at the 5th, 10th, 15th and 40th tissue culture passagelevels. At higher passage levels to the virulence of the virus for babypigs became progressively less until it was rendered avirulent.

It has been found that the tissue culture techniques of the inventiongive a high yield of virus from the tissues of swine, sheep, cattle,dogs, ferrets and cats. Other animal tissues can also be employed tosupport T .G.E. virus growth. Production of vaccine from tissues otherthan porcine, particularly canine, e.g. dogs and foxes, eliminatesundesired porcine contamination viruses. Canine tissues or which may bepresent in the cultivated canine tissues or canine tissue cells employedin the present method are not pathogenic to swine and therefore do notinfect swine to which the canine origin vaccine of the invention isadministered.

The virus produced by the invention may be diluted 4 be conferred onnursing pigs by injection of the T.G.E. vaccine of the invention intosows prior to birth of their pigs. Pigs which nurse from said vaccinatedsows are immune to T.G.E.

EXAMPLE 2 The filtrate was prepared according to Example 1 and T.G.E.virus was propagated according to the process described above using 9parts Hanks Balanced Salt Solution and 1 part inactivated horse serum.Virus-containing fluids from 10th to 12th tissue culture passages werediluted 1 to 20 and inoculated into standard Povitsky bottles containinga monolayer of dog kidney cells. The bottles were incubated at 37 C. for10 hours at which time no gross cytopathic effect was observed. Themonolayers were detached into the fluids by freezing and thawing.Identity of virus content in the fluids was determined by oralinoculation of 1 ml. doses of each serial tenfold dilution into babypigs which were housed in individual isolated cages. Virus infectivitytiters of at least l0 were observed.

When it is desired to prepare an inactivated vaccine, the viruscontaining culture is combined with a stabilizing menstrum and incubatedat 25 C., preferably for five days.

After incubation the vaccine can be freeze dried or it can be stored ata low temperature, e.g. 4 C. or 35 C. Inactivation of the virus wasconfirmed when oral administration of 10 ml. of the fluid did notproduce T.G.E. in baby pigs.

EXAMPLE 3 The effectiveness of the vaccines produced by Example 2 weretested by intramuscular inoculation of sows in their last six weeks ofgestation. All sows were from specific pathogen free stock and were fromfarms which had no T.G.E. for over 20 years. Forty-eight hours afterbirth two to three pigs (controls) from each litter were removed fromthe sow to isolation quarters. These controls, which received no sowsmilk for two days, and were therefore susceptible to T.G.E., and allpigs remaining With the sow were orally challenged at four days of agewith 1,000 infectious doses of T.G.E. Pigs farrowed by a non-vaccinatedsow (control) were also challenged and the results appear in Table I.

TABLE 1.IMMUNIZATION OF SOWS WITH INACTIVATED TISSUE CULTURE T.G.E.VIRUS Challenge results Number pigs with/ Number dead pigs T.G.E.symptoms from challenge/total total number number challenge challengedInoculum Vaccrnation- Number (nil) days before days sow Pigs withIsolated Iigs with Isolated farrowing was sick sow gs sow pigs SowNumber 1 4 0 0/5 2 2/2 0/5 2/2 2 0 0/5 1 2/2 0/5 2 4 0 0/4 2/2 0/4 2/2 20 0/5 3/3 0/5 3/3 0 Cont lsow... 2 6/6 2/2 6/6 2/2 2 2 1/6 2/2 6/6 2/2 12 2/4 2/2 4/4 2/2 5 0 2/3 1/ 1 3/3 1/1 2 1 0/9 2/2 0/5) 2/2 0 Controlsow..- 6 9/9 2/2 9/9 2/2 1 Sows 1 and 2 received non-desiccated vaccine.Sows 3, 4, 6, 7 8 and 0 received desiccated and reconstituted vaccine.

2 Presence of T.G.E. virus confirmed in other pigs by oral inoculationof bacteriologically sterile intestinal extracts from these pigs.

according to potency or it may have added thereto stabilizers or othernontoxic substances. For use as a vaccine, the virus may be desiccated,e.g. by freeze drying, or it may be prepared in liquid form.

Administration of the vaccines of the invention is practical only toswine which can produce protective antibodies. Since baby pigs areimmunologically immature at the time they are mot susceptible to T.G.E.,they cannot be successfully vaccinated. However, immunity can Sows whichwere vaccinated with 2 or 4 ml. of inactivated T.G.E. vaccine beforefarrowing conferred to their pigs a passive immunity to T.G.E. Pigs(controls) which were transferred from the sow to isolation andchallenged two days later with 1,000 infectious doses of T.G.E.generally developed watery diarrhea and vomition in 24 to 48 hours anddied within four to five days after challenge. Experimentally infectedimmune pigs nursing from the vaccinated sow showed no symptoms of T.G.E.

as long as they could obtain milk from the sow. In some cases the sow,which was not experimentally infected, developed a slight fever,diarrhea and agalactia for one or two days. As a result the pigs couldnot obtain milk from this sow and therefore developed diarrhea.Resumption of lactation in the sow induced recovery of the diarrheicpigs. Other experimental data have confirmed these observations that acontinuous supply of sows milk to the pig is necessary for protectionfrom T.G.E.

Preferably vaccination should consist of at least a 2 ml. doseadministered within six weeks of farrowing.

Non-vaccinated sows, on the other hand, did not confer immunity toT.G.E. to their pigs. These pigs developed watery diarrhea and vomitionin 24 to 48 hours and died within six to seven days after challenge. Thevaccine was also an effective immunizing agent for sows. Non-vaccinatedsows developed more severe symptoms of T.G.E. in comparison tovaccinated sows.

EXAMPLE 4 Fluids containing T.G.E. virus, propagated, as described inExample 2 for 40 passages on dog kidney, were combined with thestabilizing menstrum in a ratio of 50% fluid to 50% stabilizer andfreeze dried. The avirulence of the vaccine was tested by several oralinoculations of the equivalent of five or ten ml. of undiluted tissueculture fluids into baby pigs. No symptoms of T.G.E. were observedduring the ten day to two week post-inoculation observation period.Presence of T.G.E. virus was confirmed by electron microscopicexamination of the fluids.

EXAMPLE 5 The effectiveness of the attenuated vaccine produced byExample 4 was tested by intramuscular inoculation of sows- 17 or 34 daysbefore farrowing. These results are depicted in Table 2.

without substantial loss of its antigenicity comprising inoculatingvirulent virus derived from infected tissues of a pig into 'a firsttissue culture prepared from T.G.E.- virus-infected pig tissues, orT.G.E.-virus-growth-supporting non-porcine bovine, canine, feline,ferret or ovine tissue cultures and allowing the virus to grow thereinwhile continuing incubation, removing the viral particles from saidculture and introducing them into another such T.G.E.virus-growth-supporting porcine or non-porcine tissue culture andcontinuing incubation, inoculation and incubation of the virus from onetissue culture to another at intervals until the virus has lost itspathogenicity, and is avirulent when orally inoculated into baby pigs,and, when tested, no symptoms of T.G.E. are observed during a ten day totwo week post-inoculation observation period after several oralinoculations of the equivalent of 5 or 10 ml. of undiluted tissueculture fluids into baby pigs and the final attenuated virus culture isharvested and prepared in vaccine form by being combined with astabilizer and stored said attenuated vaccine being effective toimmunize sows and their nursing pigs against T.G.E.; without producingsymptoms of T.G.E.

2. A method according to claim 1 wherein the stabilized culture isfreeze dried prior to storing.

References Cited UNITED STATES PATENTS 3,479,430 11/ 1969 Welter 424-89FOREIGN PATENTS 669,881 3/1966 Belgium. 1,058,340 2/ 1967 Great Britain.

OTHER REFERENCES Lee, Km. Ann. N.Y. Acad. Sci. 66: 191-5 (1956). Eto, M.et al.: J. Jap. Vet. Med. Assn. 15: 16-20 (1962).

TABLE l.-IMMUNIZATION 0F SOWS WITH INACIIVATED TISSUE CULTURE T.G.E.VIRUS Challenge results Number dead pigs Number pigs with T.G.E.symptoms/ from challenge/total total number number challenge challengedVaccination- Number Inocnlum days before days sow Pigs with IsolatedPigs with Isolated (ml.) farrowing was sick sow pigs sow pigs SowNumber:

5 0 Control sow..- 2 6/6 2/2 6/6 2/2 Attenuated T.G.E. vaccineeffectively immunized sows (actively) and their baby pigs (passively).Control pigs, which were removed from the sow and challenged 48 hourslater with 1,000 infectious doses of T.G.E., developed typical symptomsof T.G.E. and died. None of the immune pigs, nursing from the vaccinatedsows, died, although one had watery diarrhea for one day. Thenonvaccinated sow was susceptible to T.G.E. and did not transferimmunity to T.G.E. to her pigs.

The methods and products of the present invention have been found to beeffective, convenient and practical for conferring immunity on nursingpigs and the sow against T.G.E.

In view of the above, it will be seen, that the objectives of theinvention are achieved and other advantageous results obtained. Asvarious changes could be made in the above methods and products withoutdeparting from the scope of the invention, it is intended that allmatter contained in the above description shall be interpreted asillustrative and not in a limiting context.

What is claimed is:

1. A method of attenuating virulent transmissible gastroenteritis virus(T.G.E.) to a non-pathogenic form SHEP K. ROSE, Primary Examiner US. Cl.X.R. -1.1; 424--89 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTIONPatent No. 3,585,108 Dated June 15, 1971 In fl Clai'ence J. Welter It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

001. 3, line 40, "tissues or" should be --viruses--.

Col. 3, line 74, "mot" should read --most--.

C01. 5, line 38, "TABLE 1." should read --TABLE 2.-";

same line, "INACTIVA'I'ED" should read --ATTENUATED--.

Signed and sealed this 21st day of December- 1971.

(SEAL) Attest:

EDWARD M.FLE'1CHER,JR. ROBERT GOTTSCHALK Attesting Officer ActingCommissioner of Patents FORM PO-105D (10-59) USCOMM-Qc 60316-P6Q U,S,GDVERNMENY FI|NT|NG OFFICE I 9" 0-35633

